Biodegradable polyphosphoester micelles act as both background-free 31P magnetic resonance imaging agents and drug nanocarriers

In vivo monitoring of polymers is crucial for drug delivery and tissue regeneration. Magnetic resonance imaging (MRI) is a whole-body imaging technique, and heteronuclear MRI allows quantitative imaging. However, MRI agents can result in environmental pollution and organ accumulation. To address this, we introduce biocompatible and biodegradable polyphosphoesters, as MRI-traceable polymers using the 31P centers in the polymer backbone. We overcome challenges in 31P MRI, including background interference and low sensitivity, by modifying the molecular environment of 31P, assembling polymers into colloids, and tailoring the polymers’ microstructure to adjust MRI-relaxation times. Specifically, gradient-type polyphosphonate-copolymers demonstrate improved MRI-relaxation times compared to homo- and block copolymers, making them suitable for imaging. We validate background-free imaging and biodegradation in vivo using Manduca sexta. Furthermore, encapsulating the potent drug PROTAC allows using these amphiphilic copolymers to simultaneously deliver drugs, enabling theranostics. This first report paves the way for polyphosphoesters as background-free MRI-traceable polymers for theranostic applications.

From the real-time NMR measurements, we calculated the reactivity ratios for Et-Pn and Ph-Pn using four different nonterminal models, as proposed by Jaacks, 1 BSL, 2 and Frey 3 (ideal integrated model) and the Meyer-Lowry 4 ( Supplementary Fig. 1). All four models resulted in similar reactivity ratios under these conditions, r(Ph-Pn)= 23.9 ± 0.6 and r(Et-Pn)= 0.040 ± 0.002, for fitting up to 40 % of the total monomer conversion (Supplementary Table 2). The determined reactivity ratios from the ideal integrated model were used to calculated the polymer fraction vs. conversion (main text, Fig. 2 B); a Monte Carlo simulation was performed as reported previously (main text, Fig. 2 C,D). 5 This visualization of the copolymerization, clearly shows the formation of a gradient structure in the final macromolecule.

Monte Carlo simulations
Monte Carlo simulations were performed in MATLAB R2020b.
First the reactivity ratios rA and rB were set to the calculated values. ƒA,0 was set to the initial fraction of monomer A. Each simulation was performed with 10 6 chains (nchains), the PDI was set to one (we set the molar mass dispersity to unity to exclude chain length effects on the visualization). The total number of monomer molecules is set to the number of chains multiplied with the degree of polymerization (DP). The final degree of polymerization was DP=100.
If F>w then add monomer A to the chain, otherwise monomer B.
If chain k has a DP>0, then: A random number w in the interval [0,1] is created. The monomer at the chain end k is called e. The reactivity ratio corresponding to this chain k was used to calculate the probability of addition of the same monomer as e: If Fe>w then add monomer equal to e, otherwise use the monomer unequal to the chain end.
Then the number of consumed monomers nx is reduced by 1. Then go to the step, where the next chain k is selected by sequence (➔ PDI=1). These steps are repeated until DP=100.   The CMC was measured using pyrene assay, as described in literature (Fig. S3). 6 The low   Table 4).

Size exclusion chromatography (SEC) of Ph-grad-Et-PPn copolymers
In the case PPnGRAD micelles, a monoexponential decay provides a good description of the measurement results for Et-PPn copolymer at time t<1 s. Yet, some deviation, in the slow relaxation region is present ( Supplementary Fig. 10 b,c, Supplementary Table 4). A biexponential fit, provides a good model through the whole measurement range ( Supplementary Fig. 10 b,c, Supplementary Table 4). Conversely, in the case of PPnSHELL a monoexponential decay did not provide a good fit of the experimental data ( Supplementary   Fig. 10 e,f, Supplementary Table 4). The results could be fit with biexponential fit at t<2 s; only the triexponential decay provided a good fit over the all measured echo times. However, shortest T2,1 is too fast to be measured accurately with the used echo times (Supplementary Table 4).
In general, the region of the hydrophilic Et-PPn chain that is closer to the hydrophobic core

Degradation of PPnGRAD micelles in vivo
Supplementary Fig. 13. Degradation of PPnGRAD in vivo in three additional M. sexta. The

Materials
Solvents and chemicals were purchased from Acros Organics, Merck / Sigma-Aldrich or Fluka in at least p.a. purity. Chemicals for synthesis were additionally dried and stored over molecular sieves (4 Å), when required for synthesis (specified further in experimental procedures).
Ultrapure water with a resistivity of 18.4 MΩ cm -1 (Milli-Q, Millipore) was used for the selfassembly experiments. 2-(Benzyloxy)ethanol was acquired from ABCR, distilled from calcium hydride and stored over molecular sieve (3 and 4 Å) and under inert gas prior to use. DBU was purchased from Sigma Aldrich, distilled from calcium hydride and stored over molecular sieves PROTAC ARV-825 was obtained from Chemietek (USA). Deuterated solvents were purchased from Deutero GmbH (Kastellaun, Germany) and Merck and used as received.

Polymerizations
Representative procedure for the ring-opening polymerization catalyzed with DBU.
Polymerization was performed according to a modified literature protocol. 9 The particular monomers were weighed in a flame-dried Schlenk-tube, dissolved in dry benzene and dried by lyophilization. The monomer was dissolved in dry dichloromethane to a total concentration

Real-time copolymerization kinetics
To study the incorporation behavior of the different monomers during the copolymerization, the same reaction mixture as used for polymerization was prepared and transferred into a dry NMR tube under inert gas. This mixture was used to setup all NMR parameters (i.e. shim and lock) at 263 K. The reaction was initiated by adding the calculated volume of catalyst solution (3 eq of DBU in respect to the initiator). The NMR tube was quickly pleased in the NMR spectrometer and the 31 P-spectra were measured at different time points to follow the polymerization. To determine the reactivity ratios, we followed the protocol of Gleede et. al.
and all methods used data from 0 up to 40 % conversion to determine reactivity values. 5

Homopolymer Dispersion
The dispersion of homopolymer was prepared using miniemulsion formulation technique similarly, as described previously. 10 Ph-PPn (20 mg) was dissolved in chloroform (0.85 g) and added to an aqueous solution of sodium dodecyl sulfate (6.9 mg) in water (3.34 g). The

PPnGRAD Micelles loaded with PROTAC ARV-825
Micelles loaded with PROTAC ARV-825 were prepared using the second approach. Gradient copolymer (10 mg) was dissolved in acetone (280 µL) and mixed with ARV-825 (Fig 14) in acetone (0.2 mg in 20 µL acetone). Subsequently, PROTAC/Polymer solution was added to ultrapure water (490 µL), forming a clear, yellow mixture. After sonication in ultrasonic bath and evaporation overnight (controlled by weighing the vial) a yellow slightly opalescent dispersion of micelles was obtained.
As a control, the procedure was repeated without adding the polymer; ARV-825 (0.2 mg in 20 mL acetone) was added to ultrapure water. The precipitation of ARV-825 was observed immediately after the addition of the acetone solution into water.

Cell Experiments
Viability assay in immune cells. To obtain circulating immune cells,